Project Summary MicroRNAs (miRs) are small non-coding RNA molecules (~22 nucleotides) known to negatively regulate gene expression through RNA silencing and post-transcriptional regulation. Importantly, miRs are increasingly recognized for the maintenance of cardiac function, modulation of cardiac excitability, and the development of disease states through their classical mechanism of finely controlling the expression of target genes. miR1 is the most predominant miR in the heart and plays a critical role for cardiovascular development and cardiac electrophysiology. In this study, we questioned whether this regulation of cardiac function was solely due to the classical mechanism of miRs or if this could also be attributed to a novel role for miR1 mediated through its physical interaction with cardiac proteins. We first performed an in vitro electron mobility shift assay (EMSA) to investigate if miR1 and miR451a, which are both highly expressed in the heart, can bind with membrane proteins extracted from neonatal and adult mouse hearts. Remarkably, we found that miR1, but not miR451a, could specifically bind with membrane proteins and identified that Kir2.1, an inward rectifier potassium channel, was one of the membrane proteins that miR1 binds to. To establish the potential biophysical consequence of this binding, we performed patch clamp recordings of inward rectifier potassium current (IK1) and delivered miR1 acutely to bypass the transcriptome regulation. Importantly, we found that the current density of IK1 was significantly suppressed by acute expression of miR1. To our knowledge, this is the first discovery of the novel and groundbreaking concept that miRs physically interact with ion channels to modulate their biophysical function. Hence the overall hypothesis of this proposal is that miR1 can also regulate cardiac excitability through direct interaction with cardiac ion channels. Therefore, we propose to investigate the mechanism of miR1-ion channel interaction with the following aims: 1. Define the physical interaction between miR1 and Kir2.1. 2. Evaluate the functional implications of the physical interaction between miR1 and Kir2.1. 3. Identify other cardiac ion channels that physically interact with and are modulated by miR1. Several approaches will be used to identify the ion channels that miR1 interacts with. Importantly, we will investigate the physiological role of this physical interaction in expression systems and in cardiomyocytes. We will study the electrophysiology of cardiomyocytes by patch-clamp with acutely-delivered miR1 to investigate how miR1 modulates cardiac electrophysiology without its transcriptome effect. Our study will provide a mechanistic understanding of how miR1 physically binds with ion channels and directly regulates their functions. We will also investigate if this physical interaction between miR1 and Kir2.1 is a general function of miRs by identifying more miR1-bound ion channels. A better understanding of the biophysical interaction between miR1 and ion-channels will help us to comprehensively recognize the biophysical dysregulation of ion channel in cardiac disease.